PROJECT SUMMARY An estimated 27 million Americans age ?25 have osteoarthritis (OA) and this number is projected to escalate to more than 65 million by 2029 at a direct cost estimated at $28.6 billion. Reducing the incidence and effects of OA through effective treatment of cartilage defects would be a significant socioeconomic benefit. As the supply of suitable cartilage grafts is unable to meet clinical demand, the development of tissue engineered osteochondral grafts with mechanically functional properties would have a significant clinical impact. Examination of engineered cartilage tissues at a multi-scale level suggests local variable ECM content at the single cell level, where cells, for example, exhibiting intense metachromatic staining for ECM are juxtaposed to others with relatively little metachromatic staining. We speculate that this intrinsic cell-to-cell variability in ECM production capacity undermines or limits the peak tissue properties attainable by the whole cell population, and may also impact engineered cartilage integrative repair potential. This proposal will test the following hypotheses: H1) Cell cycle priming leads to coordinated cell tissue elaboration capacity, thereby expediting development and peak magnitude of functional tissue properties by decreasing local spatial inhomogeneity in engineered cartilage derived from clinically-relevant chondrocytes. H2) Cell cycle priming is mediated in part by primary cilia that increase in incidence post synchronization. H3) Repair of full thickness osteochondral defects with engineered cartilage constructs derived from initially (cell cycle) synchronized chondrocytes will be superior to non-synchronized (control) chondrocytes due to the unprecedented acceleration of functional tissue development associated with cell cycle priming that leads to cartilage grafts that better approximate the cartilage associated with clinical osteochondral allografts. The corresponding aims will study human and canine chondrocytes in vitro (Specific Aim 1) and engineered canine cartilage constructs in vivo with a full-thickness ostechondral focal defect repair model in the dog (Specific Aim 2). This NIH R21 application will explore the potential for cell cycle priming as a novel platform technology for functional tissue engineering and generation of tissues with native mechanical properties in 6 weeks or less. We will determine if the functional benefits of cell synchronization on 3D cartilage tissue formation are derived from the reduction of cell-to-cell variability and homogenization of cell ECM output. While the concept of cell synchronization is well-established in cell biology, its application for engineering cartilage, as demonstrated by our preliminary data, represents an innovation.